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dc.contributor.authorPapple, Alex G.
dc.identifier.citationPapple, A.G., 2015: INVESTIGATING DUAL-TARGETED DOMAIN ANTIBODY FUSION PROTEINS IN A CANCER MODEL, Queen Mary University of London.en_US
dc.descriptionPhD Thesisen_US
dc.description.abstractDeveloping efficacious, highly specific therapeutics is an ongoing challenge. The domain antibody (dAb) fragment platform has shown promise targeting a dAbfused therapeutic moiety to a specific tissue. This investigation sought to conclude on the optimum binding affinity combination to maximise tissue-targeting specificity and localised therapeutic potency by using a panel of dAb fusion proteins with varying affinity combinations of asialoglycoprotein receptor (ASGPR)-targeting dAb mutants and human interferon (hIFN) mutants – hIFN-ASGPRdAbs. The hIFN-ASGPRdAbs were engineered, expressed, 1,4,7- triazacyclononanetriacetic acid (NOTA)-conjugated and purified with binding affinities and potencies characterised. Gallium-68 radiolabelling of the NOTA-hIFNASGPRdAbs was optimised using a high activity fraction in a 10 minute reaction at ambient temperature in pH4.4 sodium acetate. HepG2 xenograft mouse models were injected with 68Ga-NOTA-hIFN-dAbs for biodistribution analysis and PET/CT imaging, followed by mRNA expression analysis of the xenograft tissue. ASGPR binding affinities of the NOTA-hIFN-ASGPRdAbs ranged from 0.73pM to ~528nM, and their hIFN potency ranged from 6.79pM to ~3.87nM. Targeting of HepG2 cells was driven by the dAb-mediated ASGPR targeting. Ga-68 radiolabelling efficiencies up to 98.4% and specific activities up to 2.47MBq/μg were achieved. Xenograft uptake was significantly increased through dAb-mediated ASGPR targeting compared to the non-ASGPR targeted control 68Ga-NOTA-hIFNCTRLdAb, achieving 2.42%ID/g vs. 0.68%ID/g, respectively. A trend of increasing xenograft uptake correlated with increasing ASGPR binding affinity for the panel of mutants, in spite of very high murine liver uptake. Increased xenograft uptake also correlated with a more potent hIFN-mediated anti-proliferative mRNA response in the xenograft tissue. A dAb with a mid-range affinity for ASGPR and maximum affinity hIFN was concluded to be the optimum combination. These results showed that the efficacy of a dAb fusion protein can be influenced by the two intrinsic binding affinities. Hence, dAb-mediated tissue targeting of a fused therapeutic and engineered affinity synergism strategies may hold the key to novel, selectively cytotoxic biopharmaceutical drugs.
dc.description.sponsorshipQueen Mary University of London, GlaxoSmithKline (GSK) and the EPRSC.en_US
dc.publisherQueen Mary University of Londonen_US
dc.subjectcytotoxic biopharmaceutical drugs.en_US
dc.rights.holderThe copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author

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  • Theses [2958]
    Theses Awarded by Queen Mary University of London

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